Advances in Clinical Medicine
Vol.07 No.04(2017), Article ID:22310,7 pages
10.12677/ACM.2017.74039

Research Progress of Osteogenesis-Related Signaling Pathways

Lei Zhou, Minghai Wang*

Department of Orthopedics, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai

Received: Sep. 27th, 2017; accepted: Oct. 7th, 2017; published: Oct. 16th, 2017

ABSTRACT

Objective: Osteogenesis is the foundation of bone formation and key procedure of bone metabolism. In recent years, major progress was made in the molecular mechanism of osteogenesis at home and abroad. Therefore, the mechanism and research progress of osteogenesis-related signaling pathways was reviewed. Methods: Literature about ossification and osteogenesis-relate signaling pathways in recent years were reviewed and analyzed. Results: Several signaling pathways have been found osteogenesis-related, among them, BMP-SMAD, Wnt/β-Catenin, Notch, Hedgehog, MAPK and FGF signaling pathways play the leading role in bone-formation. Besides, a complex regulatory network is composed of interactions between multiple signaling pathways. However, the specific mechanism of osteogenesis-related signaling pathways is still unclear because of limited research methods. Conclusion: To make clear the mechanism of these signaling pathways respectively and their interactions is of great significance for illustrating the complete mechanism of osteogenesis.

Keywords:Bone Metabolism, Osteogenesis, Signaling Pathway

成骨分化相关信号通路的研究进展

周雷,王明海*

复旦大学附属上海市第五人民医院骨科,上海

收稿日期:2017年9月27日;录用日期:2017年10月7日;发布日期:2017年10月16日

摘 要

目的:成骨分化是骨质形成的基础,也是骨代谢的关键步骤。近年来,关于成骨分化的分子机制,国内外取得了许多突破性的进展。故围绕成骨分化相关信号通路研究进展这一要点进行综述和分析。方法:检索近几年国内外成骨分化及成骨相关信号通路研究的文献,并作总结分析。结果:发现多条信号通路参与成骨分化,其中,BMP-SMAD、Wnt/β-Catenin、Notch、Hedgehog、MAPK、FGF信号通路在成骨分化过程中最为关键。多条信号通路间存在着相互作用,构成了一个复杂的调控网络,但由于研究手段的局限,成骨分化相关信号通路的具体作用机制仍不明了。结论:若能说明这些信号通路各自发挥作用的机制及各条通路之间的相互关系,对阐明成骨分化的具体机制具有重要意义。

关键词 :骨代谢,成骨分化,信号通路

Copyright © 2017 by authors and Hans Publishers Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

1. 概述

同躯体的其他组织器官一样,骨组织也在不断地进行着细胞代谢,称为骨代谢。它可以大致分为骨形成和骨重塑两个阶段,前者在个体生长发育期起主要作用,后者则持续整个生命周期。骨重塑始于单核-巨噬细胞来源的破骨细胞吸收骨基质,随后成骨相关细胞被募集到骨吸收部位,通过形成和分泌骨基质实现重塑,以适应机体力学环境的改变或修复骨损伤 [1] 。而成骨分化是骨生成的关键步骤,即骨髓间充质干细胞经历成骨祖细胞、成骨前体细胞、成骨细胞最终分化为骨细胞的一个复杂的过程,其中涉及到多种类型细胞间和细胞内的信号传递,如信号通路、转录因子、生长因子、MicroRNA等,形成了一个完整的骨代谢调控负反馈环路 [2] 。本文就成骨分化相关信号通路研究进展这一要点展开综述。

2. 成骨相关信号通路

2.1. BMP-SMAD信号通路

骨代谢同时受多种细胞因子及生长因子的调控,骨形态发生蛋白(BMPs)便是其中之一。BMPs在骨代谢过程中起着关键性的调控作用。BMPs属于转化生长因子β (TGF-β)超家族,是一类多效性细胞因子,根据序列相似性和功能可以将BMPs分为4个亚族:1) BMP-2和BMP-4;2) BMP-5、-6、-7、-8a和-8b;3) BMP-9和BMP-10;4) BMP-3、-3b、-11、-12、-13、-14、-15和-16,其中,BMP-2、-7、-6、-9能够促进骨质形成 [3] ,而BMP-3对成骨具有负调控作用 [4] 。TGF-β超家族的成员均通过结合双受体系统—I型和II型跨膜丝氨酸/苏氨酸激酶受体(BMPR-I、BMPR-II)介导信号转导。而在膜信号传入核内的过程中,Smad信号通路发挥着主要作用。

BMP信号分子结合并激活BMPR-II,使得BMPR-I磷酸化并进一步磷酸化BMP活化型Smads (BR-Smads)的C末端DNA结合域,磷酸化的BR-Smads与共同通路型Smads (Co-Smads)结合形成异质低聚体进入核内与转录因子相互作用,共同调控目的基因的表达 [5] 。在骨髓间充质干细胞(BMSCs)中BMP通过BR-Smads直接或间接诱导Runx2的表达,BR-Smads又可以与Runx2以物理结合的方式进一步诱导成骨分化 [6] 。I-Smads则可以抑制BR-Smads和Co-Smads的作用。此外,Samds的活性还受到许多因素的调控。小C端结构域磷酸酶1、2 (SCP-1、SCP-2)介导Smads的去磷酸化可抑制后者的转录活性 [7] 。泛素连接酶1(SMURF1)能够修饰Smad1、5 [8] ,SMURF2仅介导Smad1的泛素化 [9] 。

近年来借助实验生物反应器发现机械应力引起的骨代谢与BMP-Smads信号通路有着密切的关系:在应力刺激下的细胞具有更高的BMP反应性并更早激活Smads通路 [10] ;机械负荷能减少BMP拮抗分子的表达 [11] ;应力作用可以使BMP-2、BMP-7、碱性磷酸酶(ALP) I型胶原的表达量增加 [12] 。然而,这一现象在不同种类的细胞间并不完全一致 [13] 。

2.2. Wnt/β-Catenin信号通路

Wnts是一类与卷曲蛋白受体(FZD)结合的分泌型糖蛋白。Wnt家族分泌的因子参与细胞极化、分化、迁移、增殖和生物学功能等多个细胞生理过程。Wnt蛋白可以大致分为两类:一类激活经典的Wnt信号通路,即Wnt/β-Catenin信号通路;另一类由Wnt5a激活非经典的Wnt通路,配体与FZD结合后不依赖于β-Catenin和LRP5/6。

Wnt/β-Catenin信号通路中,Wnt配体与FZD或低密度脂蛋白受体相关蛋白5/6 (LRP5/6)形成复合物。当胞外缺乏Wnt蛋白时,该复合体无法形成,此时在糖蛋白激酶-3 (GSK-3)作用下导致β-Catenin蛋白水解,胞浆及核内的β-Catenin水平降低,最终抑制Wnt/β-Catenin信号通路。在该信号通路中,GSK-3的活性受大肠腺瘤样蛋白(APC)和轴蛋白(Axin)形成的多肽复合体的调控。当Wnt分子与LRP5/6-FZD受体复合体结合,LRP5/6胞内端磷酸化而产生Axin的结合位点,Axin结合到该位点能抑制GSK-3介导的β-Catenin水解,引起β-Catenin增加并进入核内,与细胞核中的T细胞因子(TCF)/淋巴增强因子(LEF)转录复合物结合,作为转录激活因子可引起下游靶基因表达,从而发挥调控作用 [14] 。

研究证实Wnt/β-Catenin信号通路通过控制间充质干细胞、成骨细胞、破骨细胞及软骨细胞的分化和功能来调控骨代谢。间充质细胞条件性β-Catenin基因敲除鼠在骨骼发育期表现出显著减弱的成骨分化 [15] 。然而,Wnt/β-Catenin信号通路对成骨分化的影响还取决于细胞所处的发育阶段。体外激活Wnt/β-Catenin信号通路可以促进间充质干细胞的增殖,但抑制其成骨分化 [16] 。一旦间充质干细胞开始向成骨细胞系分化,Wnt/β-Catenin信号通路虽能促进细胞生长和分化,但阻碍其终末分化为成熟的成骨细胞 [17] 。这一阶段依赖的现象同样表现在人类疾病中,如骨纤维异常增殖症是由于上调的Wnt/β-Catenin信号通路引起的成骨细胞分化和成熟障碍所导致的 [16] 。

2.3. Notch信号通路

Notch信号通路在进化上高度保守,其在细胞发育、增殖、凋亡和分化均具有调控作用。Notch的受体和配体为跨膜蛋白,其发挥功能依赖于相邻细胞间的相互接触。哺乳动物拥有4种Notch受体(Notch1-4),并根据结构不同将12种配体分为四类。

当相邻细胞表面的同源配体与受体结合,Notch受体的胞外部分和跨膜部分分别经TACE、γ-分泌酶水解,引起Notch受体胞内部分(NICD)从细胞膜上脱落并移入核内。在细胞核中NICD与RBPJ、MAML相互作用,将转录抑制子转化为激活子,激活下游HES家族、HEY家族等的基因表达 [18] 。

目前已有大量研究证明Notch信号通路在成骨分化中起作用。Tezuka等发现Notch1在成骨前体细胞(MC3T3-E1)分化的早期阶段表达增高,同时,NICD过表达的MC3T3-E1细胞在成骨分化过程中钙结节的形成量显著增加;此外,外源性Notch可以诱导多能间充质细胞系(C3H10T1/2)成骨分化,抑制成脂分化 [19] 。用hMSCs重复出上述现象的同时,发现Notch下游基因Hes-1能与Runx2相互作用并增强后者作为转录激活子的活性;Notch信号通路的激活子Maml也发现能够激活骨组织Runx2的转录 [20] 。NICD转基因鼠的成骨显著增加且表现为严重的骨硬化症,其骨组织结构异常紊乱且骨钙素(OCN)的表达明显减少,提示成骨细胞存在成熟缺陷,可能是NICD与Runx2结合并抑制后者激活OCN导致成骨细胞的未成熟状态 [21] 。也有研究团队利用NICD转基因鼠得到骨量减少的表型 [22] 。总体来说,Notch信号通路在成骨分化过程中具有调控作用,但具体机制仍存在一定的争议。

2.4. Hedgehog信号通路

Hedgehog信号通路同样在进化上高度保守,在发育和内稳态方面起着重要作用。在哺乳动物,Hedgehog蛋白可分为三类:Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Desert Hedgehog (DHH)。当胞外Hedgehog蛋白与跨膜受体(PTC)结合即解除对SMO抑制并进一步使之磷酸化,SMO激活使Hedgehog通路转录效应子Ci/Gli从Cos2释放转移入细胞核,激活相应下游靶基因的表达 [23] 。

PTCh1缺陷病人及小鼠模型表现为骨量增加,PTCh1缺陷的成骨前体细胞由于与Runx2的反应性增加及GLI3抑制物产生减少而表现为成骨分化速度增快 [24] 。与此相对,GLI1缺乏小鼠的表型为骨量减少、成骨分化减弱和破骨细胞生成增加 [25] 。另一份体外实验提出SHH能上调成骨细胞系Osx的表达,促进成骨细胞产生并间接上调破骨细胞的活力,引起骨吸收增加及骨强度下降 [26] 。在骨缺损修复初期,IHH和PTC1均表达增加 [27] ;在重塑期,成骨细胞内SHH激活以调控成骨细胞增殖、分化,破骨细胞形成以及血管生成 [28] 。利用IHH/MSCs/支架材料复合物的组织工程实验结果显示骨修复加快 [29] 。可见,Hedgehog信号通路在促进成骨分化上具有显著的作用,但三个配体分别所起的作用及机制尚有待研究。

2.5. MAPK信号通路

传统的丝裂原活化蛋白激酶(MAPKs)包括三个亚家族成员:1) ERK1/2、ERK5;2) JNK1/2/3;3) p38。MAPK通路主要参与转导胞外刺激(环境压力、生长因子、细胞因子等)引起细胞生长、分化和凋亡。一旦细胞接触刺激物,MAPKK激酶(MAP3K)被激活并磷酸化MAPK激酶(MAP2K),而后磷酸化激活MAPKs [30] 。

研究发现MAPKs在骨骼发育和骨代谢中起着重要作用,大部分研究均指向p38和ERK,JNK在成骨分化中所起的作用尚有一定的争议。如通过化学抑制JNK或siRNA干扰其表达,均引起矿化减少、成骨标志物表达下降,而其活性状态下促进成骨 [31] 。但也发现在hMSCs抑制JNK后ALP的活性增加而成骨加强 [32] 。

ERK1/2均表达于成骨细胞并在骨代谢中具有相似的功能。首先通过调控骨钙素启动子激活抑制性MEK1,此时小鼠表现为颅骨和锁骨缺陷,与Runx2缺乏的表型相似。而Runx+/−的表型可以被活性MEK1恢复 [32] 。Matsushita通过ERK1/2双突变鼠证明ERK1/2在成骨分化中具有促进作用,并能够抑制软骨膜周围的软骨分化 [33] 。Runx2磷酸化被普遍认为是ERK信号通路促进成骨分化的机制:MEK1抑制物可阻滞Runx2诱导的骨钙素表达 [34] ;ERK1/2特异的丝氨酸残基(Ser301、319)与Runx2的激活能力有关 [35] 。

选择性p38抑制剂作用于成骨细胞系或原代细胞的结果显示p38在受成骨诱导性配体BMP2、Wnt蛋白、PTH激活后表现出调控成骨分化、胞外基质沉积及矿化的作用。p38被BMP激活后通过促进SMAD1磷酸化及核定位促进成骨分化 [36] 。P38通路在Wnt3a作用下可以募集间充质干细胞 [37] 。PTH通过蛋白激酶A(PKA)激活p38调控成骨细胞的功能,说明PTH是p38上游分子之一 [38] 。

2.6. FGF信号通路

成纤维细胞生长因子(FGFs)家族由22个分泌性多肽组成,能与4个高度同源的酪氨酸激酶受体(FGFR1-4)结合,引起FGFR二聚化并磷酸化自身的酪氨酸残基,以激活多个信号转导途径及下游基因,具有调控多种生长相关进程的作用,包括软骨内成骨和膜内成骨 [39] 。

目前已发现成骨细胞能表达FGF2和FGF18。内源性FGF2表达的重要性已得到广泛关注,FGF2失效会引起成骨细胞数目减少、骨量下降 [40] 。这一表型在自然生长状态下或PTH诱导下均发现与FGF2相关,提示这一分子与骨发育密切相关 [41] 。其他成员如FGF18,该基因敲除鼠表现为骨骼延迟形成 [42] 。FGF分子能激活多个成骨相关信号通路,如wnt、ERK、p38、PLCγ和PKC等,通过激活这些信号通路,FGF可以间接地调控成骨相关基因的表达 [43] 。FGFR2激活的人成骨细胞Runx2表达显著增加 [44] 。在FGFR激活的小鼠体内检测到Sox2的表达上调,并且细胞增殖速度加快 [45] 。

3. 结论与展望

成骨分化是一个涉及多步骤的复杂生理过程,目前已证实多条信号通路在这一过程中起重要的调控作用。本篇综述总结分析了在成骨分化方面主要信号通路的相关研究进展,其中多个信号分子直接或间接影响Runx2、Osx等成骨关键转录因子的表达,最终调控成骨分化。然而,目前的研究尚存在一定的局限性。成骨分化相关的多条通路相互联系相互作用,构成了一个复杂的网络,但目前的研究以单通路为主,部分多通路研究也比较局限,还没能彻底地揭示成骨分化的具体机制。因此,以生物信息学为基础的综合性基因调控网络研究将会是接下来研究的热点。随着研究的深入,有望揭示多种骨病(骨质疏松、骨硬化症等)的分子机制,并成为骨科慢病的早期特异性标志物或靶向药物的靶点。

基金项目

本研究由闵行区自然科学基金(2017MHZ15)资助。

文章引用

周 雷,王明海. 成骨分化相关信号通路的研究进展
Research Progress of Osteogenesis-Related Signaling Pathways[J]. 临床医学进展, 2017, 07(04): 235-241. http://dx.doi.org/10.12677/ACM.2017.74039

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  46. NOTES

    *通讯作者。

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